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. 1998 Oct 15;335(Pt 2):389–396. doi: 10.1042/bj3350389

Cytosolic deglycosylation process of newly synthesized glycoproteins generates oligomannosides possessing one GlcNAc residue at the reducing end.

S Duvet 1, O Labiau 1, A M Mir 1, D Kmiécik 1, S S Krag 1, A Verbert 1, R Cacan 1
PMCID: PMC1219794  PMID: 9761739

Abstract

Recent studies on the mechanism of degradation of newly synthesized glycoproteins suggest the involvement of a retrotranslocation of the glycoprotein from the lumen of the rough endoplasmic reticulum into the cytosol, where a deglycosylation process takes place. In the studies reported here, we used a glycosylation mutant of Chinese hamster ovary cells that does not synthesize mannosylphosphoryldolichol and has an increased level of soluble oligomannosides originating from glycoprotein degradation. In the presence of anisomycin, an inhibitor of protein synthesis, we observed an accumulation of glucosylated oligosaccharide-lipid donors (Glc3Man5GlcNAc2-PP-Dol), which are the precursors of the soluble neutral oligosaccharide material. Inhibition of rough endoplasmic reticulum glucosidase(s) by castanospermine led to the formation of Glc3Man5GlcNAc2(OSGn2) (in which OSGn2 is an oligomannoside possessing two GlcNAc residues at its reducing end), which was then retained in the lumen of intracellular vesicles. Thus they were protected during an 8 h chase period from the action of cytosolic chitobiase, which is responsible for the conversion of OSGn2 to oligomannosides possessing one GlcNAc residue at the reducing end (OSGn1). In contrast, when protein synthesis was maintained in the presence of castanospermine, glucosylated oligomannosides (Glc1-3Man5GlcNAc1) were recovered in cytosol. Except for monoglucosylated Man5 species, which are potential substrates for luminal calnexin and calreticulin, the pattern of oligomannosides was similar to that observed on glycoproteins. The occurrence in the cytosol of glucosylated species with one GlcNAc residue at the reducing end implies that the deglycosylation process that generates glucosylated OSGn1 from glycoproteins occurs in the cytosol.

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Selected References

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  1. Anumula K. R., Spiro R. G. Release of glucose-containing polymannose oligosaccharides during glycoprotein biosynthesis. Studies with thyroid microsomal enzymes and slices. J Biol Chem. 1983 Dec 25;258(24):15274–15282. [PubMed] [Google Scholar]
  2. Beckers C. J., Keller D. S., Balch W. E. Semi-intact cells permeable to macromolecules: use in reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex. Cell. 1987 Aug 14;50(4):523–534. doi: 10.1016/0092-8674(87)90025-0. [DOI] [PubMed] [Google Scholar]
  3. Bonifacino J. S., Lippincott-Schwartz J. Degradation of proteins within the endoplasmic reticulum. Curr Opin Cell Biol. 1991 Aug;3(4):592–600. doi: 10.1016/0955-0674(91)90028-w. [DOI] [PubMed] [Google Scholar]
  4. Bonifacino J. S. Reversal of fortune for nascent proteins. Nature. 1996 Dec 5;384(6608):405–406. doi: 10.1038/384405a0. [DOI] [PubMed] [Google Scholar]
  5. Cacan R., Dengremont C., Labiau O., Kmiécik D., Mir A. M., Verbert A. Occurrence of a cytosolic neutral chitobiase activity involved in oligomannoside degradation: a study with Madin-Darby bovine kidney (MDBK) cells. Biochem J. 1996 Jan 15;313(Pt 2):597–602. doi: 10.1042/bj3130597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cacan R., Villers C., Bélard M., Kaiden A., Krag S. S., Verbert A. Different fates of the oligosaccharide moieties of lipid intermediates. Glycobiology. 1992 Apr;2(2):127–136. doi: 10.1093/glycob/2.2.127. [DOI] [PubMed] [Google Scholar]
  7. Chapman A. E., Calhoun J. C., 4th Effects of glucose starvation and puromycin treatment on lipid-linked oligosaccharide precursors and biosynthetic enzymes in Chinese hamster ovary cells in vivo and in vitro. Arch Biochem Biophys. 1988 Jan;260(1):320–333. doi: 10.1016/0003-9861(88)90456-0. [DOI] [PubMed] [Google Scholar]
  8. Ermonval M., Cacan R., Gorgas K., Haas I. G., Verbert A., Buttin G. Differential fate of glycoproteins carrying a monoglucosylated form of truncated N-glycan in a new CHO line, MadIA214214, selected for a thermosensitive secretory defect. J Cell Sci. 1997 Feb;110(Pt 3):323–336. doi: 10.1242/jcs.110.3.323. [DOI] [PubMed] [Google Scholar]
  9. Grard T., Herman V., Saint-Pol A., Kmiecik D., Labiau O., Mir A. M., Alonso C., Verbert A., Cacan R., Michalski J. C. Oligomannosides or oligosaccharide-lipids as potential substrates for rat liver cytosolic alpha-D-mannosidase. Biochem J. 1996 Jun 15;316(Pt 3):787–792. doi: 10.1042/bj3160787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hughes E. A., Hammond C., Cresswell P. Misfolded major histocompatibility complex class I heavy chains are translocated into the cytoplasm and degraded by the proteasome. Proc Natl Acad Sci U S A. 1997 Mar 4;94(5):1896–1901. doi: 10.1073/pnas.94.5.1896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kato T., Hatanaka K., Mega T., Hase S. Purification and characterization of endo-beta-N-acetylglucosaminidase from hen oviduct. J Biochem. 1997 Dec;122(6):1167–1173. doi: 10.1093/oxfordjournals.jbchem.a021877. [DOI] [PubMed] [Google Scholar]
  12. Kmiécik D., Herman V., Stroop C. J., Michalski J. C., Mir A. M., Labiau O., Verbert A., Cacan R. Catabolism of glycan moieties of lipid intermediates leads to a single Man5GlcNAc oligosaccharide isomer: a study with permeabilized CHO cells. Glycobiology. 1995 Jul;5(5):483–494. doi: 10.1093/glycob/5.5.483. [DOI] [PubMed] [Google Scholar]
  13. Moore S. E., Bauvy C., Codogno P. Endoplasmic reticulum-to-cytosol transport of free polymannose oligosaccharides in permeabilized HepG2 cells. EMBO J. 1995 Dec 1;14(23):6034–6042. doi: 10.1002/j.1460-2075.1995.tb00292.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Pierce R. J., Spik G., Montreuil J. Cytosolic location of an endo-N-acetyl-beta-D-glucosaminidase activity in rat liver and kidney. Biochem J. 1979 Jun 15;180(3):673–676. doi: 10.1042/bj1800673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Pierce R. J., Spik G., Montreuil J. Demonstration and cytosolic location of an endo-N-acetyl-beta-D-glucosaminidase activity towards an asialo-N-acetyl-lactosaminic-type substrate in rat liver. Biochem J. 1980 Jan 1;185(1):261–264. doi: 10.1042/bj1850261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Qu D., Teckman J. H., Omura S., Perlmutter D. H. Degradation of a mutant secretory protein, alpha1-antitrypsin Z, in the endoplasmic reticulum requires proteasome activity. J Biol Chem. 1996 Sep 13;271(37):22791–22795. doi: 10.1074/jbc.271.37.22791. [DOI] [PubMed] [Google Scholar]
  17. Saint-Pol A., Bauvy C., Codogno P., Moore S. E. Transfer of free polymannose-type oligosaccharides from the cytosol to lysosomes in cultured human hepatocellular carcinoma HepG2 cells. J Cell Biol. 1997 Jan 13;136(1):45–59. doi: 10.1083/jcb.136.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Spiro M. J., Spiro R. G. Potential regulation of N-glycosylation precursor through oligosaccharide-lipid hydrolase action and glucosyltransferase-glucosidase shuttle. J Biol Chem. 1991 Mar 15;266(8):5311–5317. [PubMed] [Google Scholar]
  19. Suzuki T., Seko A., Kitajima K., Inoue Y., Inoue S. Purification and enzymatic properties of peptide:N-glycanase from C3H mouse-derived L-929 fibroblast cells. Possible widespread occurrence of post-translational remodification of proteins by N-deglycosylation. J Biol Chem. 1994 Jul 1;269(26):17611–17618. [PubMed] [Google Scholar]
  20. Turco S. J., Stetson B., Robbins P. W. Comparative rates of transfer of lipid-linked oligosaccharides to endogenous glycoprotein acceptors in vitro. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4411–4414. doi: 10.1073/pnas.74.10.4411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Vassilakos A., Michalak M., Lehrman M. A., Williams D. B. Oligosaccharide binding characteristics of the molecular chaperones calnexin and calreticulin. Biochemistry. 1998 Mar 10;37(10):3480–3490. doi: 10.1021/bi972465g. [DOI] [PubMed] [Google Scholar]
  22. Villers C., Cacan R., Mir A. M., Labiau O., Verbert A. Release of oligomannoside-type glycans as a marker of the degradation of newly synthesized glycoproteins. Biochem J. 1994 Feb 15;298(Pt 1):135–142. doi: 10.1042/bj2980135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ward C. L., Omura S., Kopito R. R. Degradation of CFTR by the ubiquitin-proteasome pathway. Cell. 1995 Oct 6;83(1):121–127. doi: 10.1016/0092-8674(95)90240-6. [DOI] [PubMed] [Google Scholar]
  24. Weng S., Spiro R. G. Demonstration of a peptide:N-glycosidase in the endoplasmic reticulum of rat liver. Biochem J. 1997 Mar 1;322(Pt 2):655–661. doi: 10.1042/bj3220655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wiertz E. J., Jones T. R., Sun L., Bogyo M., Geuze H. J., Ploegh H. L. The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol. Cell. 1996 Mar 8;84(5):769–779. doi: 10.1016/s0092-8674(00)81054-5. [DOI] [PubMed] [Google Scholar]
  26. Wiertz E. J., Tortorella D., Bogyo M., Yu J., Mothes W., Jones T. R., Rapoport T. A., Ploegh H. L. Sec61-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction. Nature. 1996 Dec 5;384(6608):432–438. doi: 10.1038/384432a0. [DOI] [PubMed] [Google Scholar]

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